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Yeast 3-phosphoinositide-dependent protein kinase-1 (PDK1) orthologs Pkh1-3 differentially regulate phosphorylation of protein kinase A (PKA) and the protein kinase B (PKB)/S6K ortholog Sch9.

ABSTRACT: Pkh1, -2, and -3 are the yeast orthologs of mammalian 3-phosphoinositide-dependent protein kinase-1 (PDK1). Although essential for viability, their functioning remains poorly understood. Sch9, the yeast protein kinase B and/or S6K ortholog, has been identified as one of their targets. We now have shown that in vitro interaction of Pkh1 and Sch9 depends on the hydrophobic PDK1-interacting fragment pocket in Pkh1 and requires the complementary hydrophobic motif in Sch9. We demonstrated that Pkh1 phosphorylates Sch9 both in vitro and in vivo on its PDK1 site and that this phosphorylation is essential for a wild type cell size. In vivo phosphorylation on this site disappeared during nitrogen deprivation and rapidly increased again upon nitrogen resupplementation. In addition, we have shown here for the first time that the PDK1 site in protein kinase A is phosphorylated by Pkh1 in vitro, that this phosphorylation is Pkh-dependent in vivo and occurs during or shortly after synthesis of the protein kinase A catalytic subunits. Mutagenesis of the PDK1 site in Tpk1 abolished binding of the regulatory subunit and cAMP dependence. As opposed to PDK1 site phosphorylation of Sch9, phosphorylation of the PDK1 site in Tpk1 was not regulated by nitrogen availability. These results bring new insight into the control and prevalence of PDK1 site phosphorylation in yeast by Pkh protein kinases.

SUBMITTER: Voordeckers K

PROVIDER: S-EPMC3121346 | biostudies-literature | 2011 Jun

REPOSITORIES: biostudies-literature

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Yeast 3-phosphoinositide-dependent protein kinase-1 (PDK1) orthologs Pkh1-3 differentially regulate phosphorylation of protein kinase A (PKA) and the protein kinase B (PKB)/S6K ortholog Sch9.

Voordeckers Karin K Kimpe Marlies M Haesendonckx Steven S Louwet Wendy W Versele Matthias M Thevelein Johan M JM

The Journal of biological chemistry 20110429 25

Pkh1, -2, and -3 are the yeast orthologs of mammalian 3-phosphoinositide-dependent protein kinase-1 (PDK1). Although essential for viability, their functioning remains poorly understood. Sch9, the yeast protein kinase B and/or S6K ortholog, has been identified as one of their targets. We now have shown that in vitro interaction of Pkh1 and Sch9 depends on the hydrophobic PDK1-interacting fragment pocket in Pkh1 and requires the complementary hydrophobic motif in Sch9. We demonstrated that Pkh1 p ...[more]

PMID: 21531713

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Project description:The PtdIns(3,4,5)P3-dependent activation of protein kinase B (PKB) by 3-phosphoinositide-dependent protein kinases-1 and -2 (PDK1 and PDK2 respectively) is a key event in mediating the effects of signals that activate PtdIns 3-kinase. The catalytic domain of serum- and glucocorticoid-regulated protein kinase (SGK) is 54% identical with that of PKB and, although lacking the PtdIns(3,4, 5)P3-binding pleckstrin-homology domain, SGK retains the residues that are phosphorylated by PDK1 and PDK2, which are Thr256 and Ser422 in SGK. Here we show that PDK1 activates SGK in vitro by phosphorylating Thr256. We also show that, in response to insulin-like growth factor-1 (IGF-1) or hydrogen peroxide, transfected SGK is activated in 293 cells via a PtdIns 3-kinase-dependent pathway that involves the phosphorylation of Thr256 and Ser422. The activation of SGK by PDK1 in vitro is unaffected by PtdIns(3,4,5)P3, abolished by the mutation of Ser422 to Ala, and greatly potentiated by mutation of Ser422 to Asp (although this mutation does not activate SGK itself). Consistent with these findings, the Ser422Asp mutant of SGK is activated by phosphorylation (probably at Thr256) in unstimulated 293 cells, and activation is unaffected by inhibitors of PtdIns 3-kinase. Our results are consistent with a model in which activation of SGK by IGF-1 or hydrogen peroxide is initiated by a PtdIns(3,4, 5)P3-dependent activation of PDK2, which phosphorylates Ser422. This is followed by the PtdIns(3,4,5)P3-independent phosphorylation at Thr256 that activates SGK, and is catalysed by PDK1. Like PKB, SGK preferentially phosphorylates serine and threonine residues that lie in Arg-Xaa-Arg-Xaa-Xaa-Ser/Thr motifs, and SGK and PKB inactivate glycogen synthase kinase-3 similarly in vitro and in co-transfection experiments. These findings raise the possibility that some physiological roles ascribed to PKB on the basis of the overexpression of constitutively active PKB mutants might be mediated by SGK.

Project description:The yeast proteins Pkh1 and Pkh2 are functional counterparts of the mammalian 3-phosphoinositide-dependent protein kinase 1 (PDK1). Cells carrying simultaneous deletion in both genes, PKH1 and PKH2 are not viable, at least in some genetic backgrounds (Casamayor et al., 1999; Inagaki et al., 1999). Consequently, a different strategy is needed to study the function of these apparently redundant gene products. The approach that has been successfully used to dissect the functions of these proteins consist in the use of a strain that combine the disruption of PKH2 with the temperature sensitive pkh1D398G allele (Inagaki et al., 1999). Since Pkh1 and Pkh2 phosphorylate Pkc1 in vitro in its activation loop (Thr983), and the cell lysis defects of the pkh1D398G pkh2 strain at the restrictive temperature are partially restored by constitutive activation of the Pkc1-Slt2 MAPK pathway, it has been suggested that the Pkh activity is important for maintenance of the cell wall integrity in S. cerevisiae (Inagaki et al., 1999; Friant et al., 2001). We considered it necessary that a different genetic strategy would be convenient in order to carry out specific studies about the functions of the Pkh proteins. In order to avoid incubation at temperatures that activate the Slt2 pathway, and induce cellular lysis, we decided to use a strategy based on the use of a regulatable promoter. We replaced 487 bp located immediately upstream the start PKH2 codon by a cassette containing the doxycycline-repressed promoter tetO7. In this strain (MB002) the PKH2 expression could be switched off by addition of doxycycline. In order to deplete the cell for the Pkh activity, we disrupted both, the PKH1 and PKH3 coding regions, obtaining the new SDP8 strain. Our results show that the long term depletion of Pkh results in a cell transcriptional response that include overexpression of genes typically induced in response to oxidative stress, as well as up-regulation of chaperons and genes that induce the unfolded protein response.

Dataset Information

Yeast 3-phosphoinositide-dependent protein kinase-1 (PDK1) orthologs Pkh1-3 differentially regulate phosphorylation of protein kinase A (PKA) and the protein kinase B (PKB)/S6K ortholog Sch9.

Publications

Yeast 3-phosphoinositide-dependent protein kinase-1 (PDK1) orthologs Pkh1-3 differentially regulate phosphorylation of protein kinase A (PKA) and the protein kinase B (PKB)/S6K ortholog Sch9.

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